The statements in the Background are not necessarily meant to endorse the characterization in the cited references.
Telomeres, the tips of eukaryotic chromosomes, protect the chromosomes from nucleolytic degradation, end-to-end fusion, and recombination. Telomeres are structures at the ends of chromosomes characterized by repeats of the nucleotide sequence (5′-TTAGGG-3′)n. Telomeres shorten as a consequence of normal cell division and critically short telomeres lead to cellular senescence or apoptosis. A rich body of epidemiological and clinical studies in humans in the past decade has linked short telomere length to high risks of aging-related disease and all-cause mortality (Puterman, E. and E. Epel, Soc Personal Psychol Compass, 2012. 6(11) 807-825; Zhu, H., M. Belcher, and P. van der Harst, Clin Sci (Lond), 2011. 120(10) 427-40; and Fyhrquist, F. and O. Saijonmaa. Ann Med, 2012. 44 Suppl 1 S138-42). Genetic, environment, lifestyle, and behavioral factors collectively impact telomere length. Therefore, telomere length has become an index for overall health, disease, and mortality risk.
While average telomere length was measured in almost all the clinical studies published and has demonstrated utility in stratifying patient disease and mortality risk, recent work in mice has also shown that the population of short telomeres is the triggering signal to senescence or apoptosis (Hemann, M. T., et al. Cell, 2001. 107(1) 67-77), and thus disease and mortality risk. In a study reported by Hemann et al, 6th generation telomerase RNA knockout mice (mTR−/− G6) with short telomeres were crossed with mice heterozygous for telomerase (mTR+/−) with long telomeres. The phenotype of the telomerase null offspring mirrors that of the mTR−/− parent despite the fact that half of their telomeres are long, suggesting that the quantity of short telomeres, and not average telomere length, is critical for cell viability and chromosome stability. In people taking a natural product-derived telomerase activator (TA-65®), a significant reduction in the percentage of short (<3 or <4 kbp) telomeres (as measured by a quantitative FISH technology; see (Canela, A., et al. Proc Natl Acad Sci USA, 2007. 104(13) 5300-5) was detected in the leukocytes, although no change in mean telomere length was seen (Harley, C. B., et al., Rejuvenation Res. 2011. 14(1) 45-56). Changes in the percentage of short telomere abundance therefore is expected to be a more sensitive measurement of the effects of lifestyle and pharmacological or other interventions on telomeres. Another study (Vera et al., “The Rate of Increase of Short Telomeres Predicts Longevity in Mammals”, Cell Reports (2012), world wide web URL: dx.doi.org/10.1016/.celrep.2012.08.023) found that “the rate of increase in the abundance of short telomeres was a predictor of lifespan”.
Various methods have been developed for the measurement of telomere length, including Southern blotting (Kimura, M. et al., Nature Protocols, 2010, 5:1596-1607), Q-FISH (Rufer, N. et al., Nat. Biotechnol., 1998, 16:743-747), flow FISH (Baerlocher, G. M. et al., Cytometry, 2002, 47:89-99), a higher throughput modification of the Q-FISH assay (HTQ-FISH; see Canela, A. et al., PNAS, 2007, 104: 5300-5305), dual-label centromeres and telomeres FISH (Cen/Tel FISH) (Vander Griend D. J., et al. Prostate 2009 Oct. 1; 69(14):1557-64. doi: 10.1002/pros.21001), dot blot (Kimura M, Aviv A. 2011 NAR), and qPCR (Cawthon, R. M., Nucleic. Acids Res., 2002, 30(10):e47; and Cawthon R M. Nucleic Acids Res. 2009, 37(3):e21).
q-PCR-telomere length (qPCR-TL) measures the abundance of average telomeres normalized with a single copy gene, expressed as T/S ratios. To convert T/S ratios to absolute length in number of bp, telomere restriction fragment length (TRF) various methods have been reported. For example, it was previously reported that this conversion could be determined by Southern blot analysis and compared to T/S ratios (Cawthon, ibid). A linear regression formula was obtained and used to calculate the TRF length of an unknown sample based on its T/S ratio. One critical issue with this conversion is that TRF contains a region of non-telomeric sequence at its centromeric end (subtelomeric sequence). Because the length of subtelomeric sequence varies among individuals, the converted by from T/S ratios based on TRF is only an approximation.
Thus, despite advances in materials and methods for facile determination of relative telomere length or abundance, there remains a need for improved methods and materials for determining differences in telomere length or abundance in subjects compared to appropriate control populations. In particular, there remains a need to determine with great accuracy differences in the relative telomere length or abundance in a subject in order to improve clinical assessments and/or therapeutic regimens in those same subjects. These needs and other needs are addressed by the present invention.